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Topic:Biophysics

Biophysics and horses examines the intersection of physical principles and biological processes in equine systems. This field applies concepts from physics to understand the mechanics, structure, and function of horses at various levels, from cellular to organismal. Research in equine biophysics includes studies on muscle dynamics, locomotion, and the biomechanics of movement, as well as investigations into the physical properties of equine tissues and their responses to external forces. This page compiles peer-reviewed research studies and scholarly articles that explore the application of biophysical methods and theories to enhance the understanding of equine biology and physiology.
Equine pituitary pars intermedia dysfunction: a spontaneous model of synucleinopathy.
Scientific reports    August 6, 2021   Volume 11, Issue 1 16036 doi: 10.1038/s41598-021-95396-7
Fortin JS, Hetak AA, Duggan KE, Burglass CM, Penticoff HB, Schott HC.Equine pituitary pars intermedia dysfunction (PPID) is a common endocrine disease of aged horses that shows a similar pathophysiology as Parkinson's Disease (PD) with increased levels of α-synuclein (α-syn). While α-syn is thought to play a pathogenic role in horses with PPID, it is unclear if α-syn is also misfolded in the pars intermedia and could similarly promote self-aggregation and propagation. Consequently, α-syn was isolated from the pars intermedia from groups of healthy young and aged horses, and aged PPID-afflicted horses. Seeding experiments confirmed the prion-like properties...
The examination of biophysical parameters of the skin in Polish Konik horses.
PloS one    June 21, 2021   Volume 16, Issue 6 e0250329 doi: 10.1371/journal.pone.0250329
Cekiera A, Popiel J, Siemieniuch M, Jaworski Z, Slowikowska M, Siwinska N, Zak A, Niedzwiedz A.This study aimed to assess the biophysical parameters of the skin in Polish Konik horses (Polish primitive horses). According to the authors, this is the first assessment performed on such a wide scale in this group of animals. The evaluation carried out is innovative both with regards to the breed of the animals and the wide scope of the physicochemical skin assessment. The study group comprised mares, stallions and geldings, and the evaluations concerned transepidermal water loss, corneometry, pH, skin temperature assessment and mexametry. These parameters were assessed in five skin regions:...
The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis.
Animals : an open access journal from MDPI    March 9, 2021   Volume 11, Issue 3 doi: 10.3390/ani11030745
Martin de Bustamante M, Gomez D, MacNicol J, Hamor R, Plummer C.The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relativ...
Experimental and theoretical studies into the release of blood droplets from weapon tips.
Forensic science international    August 22, 2019   Volume 303 109934 doi: 10.1016/j.forsciint.2019.109934
Adam CD.The formation and release under gravity of blood droplets from simulated weapon tips has been investigated experimentally and the results analysed and interpreted using established theoretical models for detached pendent drops. Droplets were produced from a series of conical nozzles, manufactured with a range of cone angles and including a set of un-bored conical tips, under conditions where the tip was either non-wetted or pre-wetted with blood. For the former, radius-limited case, detached droplet volumes were found to agree well with the predictions of both the pendent drop weight and drop ...
[Quality Assessment of Bovine and Equine Colostrum – An Overview].
Schweizer Archiv fur Tierheilkunde    May 9, 2019   Volume 161, Issue 5 287-297 doi: 10.17236/sat00205
Schneider F, Wehrend A.Good quality colostrum intake is essential for passive immunity in neonatal calf and foals. The quality of colostrum depends on its IgG content. Since in practice the possibilities for a direct determination of these parameter are limited, an estimation of colostrum quality is often based on its biophysical properties. The simple methodology allows to perform measurements directly on site and immediately after birth. The biophysical properties included in the current review are density, refractive index, relative density, viscosity, pH, and color. The determination of the density of bovine col...
Aging does not change the compressive stiffness of mandibular condylar cartilage in horses.
Osteoarthritis and cartilage    August 23, 2018   Volume 26, Issue 12 1744-1752 doi: 10.1016/j.joca.2018.08.007
Mirahmadi F, Koolstra JH, Fazaeli S, Lobbezoo F, van Lenthe GH, Snabel J, Stoop R, Arbabi V, Weinans H, Everts V.Aging can cause an increase in the stiffness of hyaline cartilage as a consequence of increased protein crosslinks. By induction of crosslinking, a reduction in the diffusion of solutions into the hyaline cartilage has been observed. However, there is a lack of knowledge about the effects of aging on the biophysical and biochemical properties of the temporomandibular joint (TMJ) cartilage. Hence, the aim of this study was to examine the biophysical properties (thickness, stiffness, and diffusion) of the TMJ condylar cartilage of horses of different ages and their correlation with biochemical p...
Influence of hair clipping on transepidermal water loss values in horses: a pilot study.
Polish journal of veterinary sciences    April 7, 2018   Volume 21, Issue 1 35-38 doi: 10.24425/119019
Szczepanik MP, Wilkołek PM, Adamek ŁR, Gołyński M, Sitkowski W, Taszkun I.The measurement of transepidermal water loss (TEWL) is one of the biophysical skin parameters used to assess skin barrier function. Assessment of transepidermal water loss, may depend on such factors as body region, age, sex or breed and the hair coat has been considered as one of the factors that may cause variation of TEWL values. The aim of our research was an examination of the influence of clipping on the amount of TEWL. The examination was performed with 12 Wielkopolska horses with Courage Khazaka Multi Probe Adapter 5 and a TEWL TM 300 probe. The TEWL values were statistically constant ...
Progress in cell-based therapies for tendon repair.
Advanced drug delivery reviews    December 24, 2014   Volume 84 240-256 doi: 10.1016/j.addr.2014.11.023
Gaspar D, Spanoudes K, Holladay C, Pandit A, Zeugolis D.The last decade has seen significant developments in cell therapies, based on permanently differentiated, reprogrammed or engineered stem cells, for tendon injuries and degenerative conditions. In vitro studies assess the influence of biophysical, biochemical and biological signals on tenogenic phenotype maintenance and/or differentiation towards tenogenic lineage. However, the ideal culture environment has yet to be identified due to the lack of standardised experimental setup and readout system. Bone marrow mesenchymal stem cells and tenocytes/dermal fibroblasts appear to be the cell populat...
The past, present and future in scaffold-based tendon treatments.
Advanced drug delivery reviews    December 10, 2014   Volume 84 257-277 doi: 10.1016/j.addr.2014.11.022
Lomas AJ, Ryan CN, Sorushanova A, Shologu N, Sideri AI, Tsioli V, Fthenakis GC, Tzora A, Skoufos I, Quinlan LR, O'Laighin G, Mullen AM, Kelly JL....Tendon injuries represent a significant clinical burden on healthcare systems worldwide. As the human population ages and the life expectancy increases, tendon injuries will become more prevalent, especially among young individuals with long life ahead of them. Advancements in engineering, chemistry and biology have made available an array of three-dimensional scaffold-based intervention strategies, natural or synthetic in origin. Further, functionalisation strategies, based on biophysical, biochemical and biological cues, offer control over cellular functions; localisation and sustained relea...
Sperm trajectories form chiral ribbons.
Scientific reports    April 17, 2013   Volume 3 1664 doi: 10.1038/srep01664
Su TW, Choi I, Feng J, Huang K, McLeod E, Ozcan A.We report the discovery of an entirely new three-dimensional (3D) swimming pattern observed in human and horse sperms. This motion is in the form of 'chiral ribbons', where the planar swing of the sperm head occurs on an osculating plane creating in some cases a helical ribbon and in some others a twisted ribbon. The latter, i.e., the twisted ribbon trajectory, also defines a minimal surface, exhibiting zero mean curvature for all the points on its surface. These chiral ribbon swimming patterns cannot be represented or understood by already known patterns of sperms or other micro-swimmers. The...
Dielectric properties of dipicrylamine-doped erythrocytes, cultured cells and lipid vesicles.
Bioelectrochemistry (Amsterdam, Netherlands)    March 4, 2013   Volume 92 14-21 doi: 10.1016/j.bioelechem.2013.02.003
Asami K.Horse erythrocytes, murine lymphoblasts (L5178Y) and lipid vesicles that were treated with dipicrylamine (DPA) as a lipophilic ion were studied by dielectric spectroscopy over a frequency range of 10 Hz to 10 MHz. The DPA-treated cells and lipid vesicles showed low-frequency (LF) dielectric dispersion around 1-10 kHz in addition to β-dispersion due to the Maxwell-Wagner effect. The LF dispersion corresponds to that found in previous electrorotation (ROT) studies on DPA-treated cells, being due to the translocation of mobile ions in the plasma membranes. Analysis of the LF dispersion based on ...
Membrane phase behavior during cooling of stallion sperm and its correlation with freezability.
Molecular membrane biology    April 5, 2012   Volume 29, Issue 3-4 95-106 doi: 10.3109/09687688.2012.674161
Oldenhof H, Friedel K, Akhoondi M, Gojowsky M, Wolkers WF, Sieme H.Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures...
Thermally-mediated ultrasound-induced contraction of equine muscular arteries in vitro and an investigation of the associated cellular mechanisms.
Ultrasound in medicine & biology    November 21, 2011   Volume 38, Issue 1 152-161 doi: 10.1016/j.ultrasmedbio.2011.10.017
Martin EM, Duck FA, Winlove CP.We have previously shown that MHz frequency ultrasound causes contraction of the carotid artery in vitro. We now extend this investigation to equine mesenteric arteries and investigate the cellular mechanisms. In vitro exposure of the large lateral cecal mesenteric artery to 4-min periods of 3.2 MHz continuous wave ultrasound at acoustic powers up to 145 mW induced reversible repeatable contraction. The magnitude of the response was linearly dependent on acoustic power and, at 145 mW, the mean increase in wall stress was 0.020 ± 0.017 mN/mm(2) (n = 34). These results are consistent with our p...
Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy.
Cryobiology    June 8, 2010   Volume 61, Issue 1 115-122 doi: 10.1016/j.cryobiol.2010.06.002
Oldenhof H, Friedel K, Sieme H, Glasmacher B, Wolkers WF.Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of free...
Latherin: a surfactant protein of horse sweat and saliva.
PloS one    May 29, 2009   Volume 4, Issue 5 e5726 doi: 10.1371/journal.pone.0005726
McDonald RE, Fleming RI, Beeley JG, Bovell DL, Lu JR, Zhao X, Cooper A, Kennedy MW.Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (< or = 1 mg ml(-1)), and therefore probably acts as a wettin...
Lung surfactant function and composition in neonatal foals and adult horses.
Journal of veterinary internal medicine    December 26, 2006   Volume 20, Issue 6 1402-1407 doi: 10.1892/0891-6640(2006)20[1402:lsfaci]2.0.co;2
Christmann U, Livesey LC, Taintor JS, Waldridge BM, Schumacher J, Grier BL, Hite RD.Lung surfactant function and composition are varied and adapted to the specific respiratory physiology of all mammalian species. Objective: Lung surfactant function and composition are different in neonatal foals as compared to adult horses. Methods: Six adult horses, 7 term foals (<24 hours old), and 4 premature foals were used. Animals were part of the Auburn University teaching herd except for 3 client-owned premature foals. Methods: Bronchoalveolar lavage fluid (BALF) was obtained from all animals. Ultracentrifugation of cell-free BALF separated surfactant into crude surfactant pellets ...
Evidence for functional ATP-sensitive (K(ATP)) potassium channels in human and equine articular chondrocytes.
Osteoarthritis and cartilage    August 7, 2006   Volume 15, Issue 1 1-8 doi: 10.1016/j.joca.2006.06.017
Mobasheri A, Gent TC, Nash AI, Womack MD, Moskaluk CA, Barrett-Jolley R.Chondrocytes are highly sensitive to variations in extracellular glucose and oxygen levels in the extracellular matrix. As such, they must possess a number of mechanisms to detect and respond to alterations in the metabolic state of cartilage. In other organs such as the pancreas, heart and brain, such detection is partly mediated by a family of potassium channels known as K(ATP) (adenosine 5'-triphosphate-sensitive potassium) channels. Here we investigate whether chondrocytes too express functional K(ATP) channels, which might, potentially, serve to couple metabolic state with cell activity. ...
Miscibility of binary monolayers at the air-water interface and interaction of protein with immobilized monolayers by surface plasmon resonance technique.
Langmuir : the ACS journal of surfaces and colloids    June 28, 2006   Volume 22, Issue 14 6195-6202 doi: 10.1021/la0605642
Wang Y, Du X.The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon re...
The redox couple of the cytochrome c cyanide complex: the contribution of heme iron ligation to the structural stability, chemical reactivity, and physiological behavior of horse cytochrome c.
Protein science : a publication of the Protein Society    January 26, 2006   Volume 15, Issue 2 234-241 doi: 10.1110/ps.051825906
Schejter A, Ryan MD, Blizzard ER, Zhang C, Margoliash E, Feinberg BA.Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(...
A non-invasive method of tendon force measurement.
Journal of biomechanics    August 9, 2005   Volume 38, Issue 10 2124-2129 doi: 10.1016/j.jbiomech.2004.09.012
Pourcelot P, Defontaine M, Ravary B, Lemâtre M, Crevier-Denoix N.The ability to measure the forces exerted in vivo on tendons and, consequently, the forces produced by muscles on tendons, offers a unique opportunity to investigate questions in disciplines as varied as physiology, biomechanics, orthopaedics and neuroscience. Until now, tendon loads could be assessed directly only by means of invasive sensors implanted within or attached to these collagenous structures. This study shows that the forces acting on tendons can be measured, in a non-invasive way, from the analysis of the propagation of an acoustic wave. Using the equine superficial digital flexor...
Quantitative analysis of voltage-gated potassium currents from primary equine (Equus caballus) and elephant (Loxodonta africana) articular chondrocytes.
American journal of physiology. Regulatory, integrative and comparative physiology    March 31, 2005   Volume 289, Issue 1 R172-R180 doi: 10.1152/ajpregu.00710.2004
Mobasheri A, Gent TC, Womack MD, Carter SD, Clegg PD, Barrett-Jolley R.In this comparative study, we have established in vitro models of equine and elephant articular chondrocytes, examined their basic morphology, and characterized the biophysical properties of their primary voltage-gated potassium channel (Kv) currents. Using whole cell patch-clamp electrophysiological recording from first-expansion and first-passage cells, we measured a maximum Kv conductance of 0.15 +/- 0.04 pS/pF (n = 10) in equine chondrocytes, whereas that in elephant chondrocytes was significantly larger (0.8 +/- 0.4 pS/pF, n = 4, P </= 0.05). Steady-state activation parameters of eleph...
A comparison of solution conformation and hydrodynamic properties of equine, porcine and rabbit serum albumin using viscometric measurements.
Biochimica et biophysica acta    January 5, 2005   Volume 1748, Issue 1 100-109 doi: 10.1016/j.bbapap.2004.12.008
Monkos K.This paper presents the results of viscosity determinations on aqueous solutions of equine, porcine and rabbit serum albumin over a wide range of concentrations and at temperatures ranging from 5 degrees C to (42-45) degrees C. The results are compared with human and bovine serum albumin previously studied. Viscosity-temperature dependence is discussed on the basis of the modified Arrhenius formula. The effective specific volume, the activation energy and entropy of viscous flow for all investigated albumins are compared. Viscosity-concentration dependence, in turn, is discussed on the basis o...
Nicoletella semolina gen. nov., sp. nov., a new member of Pasteurellaceae isolated from horses with airway disease.
Journal of clinical microbiology    December 8, 2004   Volume 42, Issue 12 5542-5548 doi: 10.1128/JCM.42.12.5542-5548.2004
Kuhnert P, Korczak B, Falsen E, Straub R, Hoops A, Boerlin P, Frey J, Mutters R.Gram-negative, nonmotile bacteria that are catalase, oxidase, and urease positive are regularly isolated from the airways of horses with clinical signs of respiratory disease. On the basis of the findings by a polyphasic approach, we propose that these strains be classified as Nicoletella semolina gen. nov, sp. nov., a new member of the family Pasteurellaceae. N. semolina reduces nitrate to nitrite but is otherwise biochemically inert; this includes the lack of an ability to ferment glucose and other sugars. Growth is fastidious, and the isolates have a distinctive colony morphology, with the ...
Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants.
Biochemistry    February 13, 2003   Volume 42, Issue 6 1684-1695 doi: 10.1021/bi0271042
Qureshi SH, Moza B, Yadav S, Ahmad F.The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation X (intermediate) conformation and the second denaturation phase, X conformation D (denatured) conformation are...
Biochemical characterization and surfactant properties of horse allergens.
European journal of biochemistry    May 19, 2001   Volume 268, Issue 10 3126-3136 doi: 10.1046/j.1432-1327.2001.02217.x
Goubran Botros H, Poncet P, Rabillon J, Fontaine T, Laval JM, David B.A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri-antennary tri-sialylated N-linked glycan. Linkages of terminal N-acetylneuraminic acid to galactose were: alpha-(2-->6) in Equ c 4, and both alpha-(2...
Biophysical and 1H magnetic resonance spectroscopy characteristics of fractionated stallion ejaculates.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 101-110 
Magistrini M, Lindeberg H, Koskinen E, Beau P, Seguin F.The composition of seminal plasma must be determined to assess the possible roles of sex gland secretions in survival of stallion spermatozoa. In the present study, an automated semen collection device and 1H magnetic resonance spectroscopy were used to analyse and compare the composition of seminal plasma from fractionated and nonfractionated stallion ejaculates. The contribution of each semen component to the ejaculate (sequence of production of component and concentration) was evaluated and its relationship to biophysical parameters was determined. 1H magnetic resonance spectroscopy was use...
Articular Cartilage Optical Properties in the Spectral Range 300-850 nm.
Journal of biomedical optics    July 1, 1998   Volume 3, Issue 3 326-333 doi: 10.1117/1.429893
Ebert DW, Roberts C, Farrar SK, Johnston WM, Litsky AS, Bertone AL.Measurements of absolute total reflectance were recorded from weight-bearing (n=9) and nonweight-bearing (n=9) equine articular cartilage specimens from 300 to 850 nm using a spectrophotometer with integrating sphere attachment. Following correction of measured spectra for interfacial reflections and edge losses, Kubelka-Munk theory was applied to estimate absorption and scattering coefficient, one-dimensional light intensity distribution, and light penetration depth. Kubelka-Munk absorption coefficients ranged from ∼7 cm-1 at 330 nm to ∼1 cm-1 at 850 nm. A localized absorption peak wa...
Interaction of horse heart cytochrome c with lipid bilayer membranes: effects on redox potentials.
Journal of bioenergetics and biomembranes    June 1, 1997   Volume 29, Issue 3 211-221 doi: 10.1023/a:1022401825287
Salamon Z, Tollin G.Cyclic voltammetry has been used to study the effects of interactions between horse cytochrome c and solid-supported planar lipid membranes, comprised of either egg phosphatidylcholine (PC) or PC plus 20 mol.% cardiolipin (CL), on the redox potential and the electrochemical electron transfer rate between the protein and a semiconductor electrode. Experiments were performed over a wide range of cytochrome c concentrations (0-440 microM) at low (20 mM) and medium (160 mM) ionic strengths. Three types of electrochemical behavior were observed, which varied as a function of the experimental condit...
Molecular diffusion into horse spleen ferritin: a nitroxide radical spin probe study.
Biophysical journal    September 1, 1996   Volume 71, Issue 3 1587-1595 doi: 10.1016/S0006-3495(96)79361-X
Yang X, Chasteen ND.Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike th...
Influence of glycerol on the structure and stability of ferric horse heart myoglobin: a SAXS and circular dichroism study.
Biochimica et biophysica acta    June 7, 1996   Volume 1295, Issue 1 51-58 doi: 10.1016/0167-4838(96)00010-6
Barteri M, Gaudiano MC, Santucci R.The influence of glycerol on the structural properties of Fe(III)-horse heart myoglobin has been investigated by absorbance, CD and SR-SAXS spectroscopy. The results obtained indicate that both the tertiary and the secondary (alpha-helix) conformations of the protein are influenced by glycerol; in particular, an increase of approx. 8% in helical content was observed. Further, analysis of both the acid- and guanidine-induced denaturation transitions points to a glycerol-induced decreased stability of the tertiary structure; conversely, the alpha-helix conformation is found to be stabilized by t...